Reconstitution of microtubule nucleation in vitro reveals novel roles for Mzt1

Protein-protein interaction within the MGM holocomplex has been investigated by chemical cross-linking mass spectrometry using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) cross-linker. Although this analysis was not exhaustive, we observed crosslinks between Alp4 and Alp6 along the length of these two proteins, consistent with their general parallel lateral alignment in current models for gamma-TuC organization. In addition, we observed specific cross-links from both Alp4 and Alp6 to the Mto1 [bonsai] CM1 domain and/or its immediate flanking regions. Interestingly, crosslinks from Alp4 and Alp6 N-terminal regions tended to be to the C-terminal portion of the CM1 domain, while crosslinks from Alp4 and Alp6 C-terminal regions tended to be to the N-terminal portion of the CM1 domain. This raises the possibility that the CM1 domain, which is adjacent to coiled-coil regions, may be oriented antiparallel to Alp4 and Alp6.